Project Summary Little is known about the role that extracellular vesicles (EVs) play in physiology and disease. EVs of various sizes and origins are able to transport lipids, proteins, and various RNA species. Moreover, EVs are implicated in the pathophysiology of multiple disorders including cancer, arthritis, and several neurodegenerative diseases. In particular, EVs from microglia, the immune cell of the brain, are able to spread alpha synuclein in Parkinson?s disease, amyloid beta in AD, and recently, exosomes, small EVs derived from exocytosis of multivesicular bodies (MVBs), have been shown to propagate pathogenic tau and worsen pathology in the PS19 mouse model of tauopathy. One protein which may provide insight into the secretion of EVs is TMEM16F, a Ca2+-activated ion channel that is required for lipid scramblase activity to move lipids bidirectionally across the lipid bilayer. Knockout of TMEM16F has been shown to reduce phosphatidylserine exposure and decrease microvesicles (MVs), EVs formed by direct outward budding of plasma membrane, from platelets, erythrocytes, macrophages, and neutrophils. The goal of my research is to study how TMEM16F affects microglial EVs and neurodegeneration. In addition to fewer MVs, I have found knockout of TMEM16F produces more exosomes, possibly through increased fusion of MVBs to the plasma membrane. To assess if this increase would exacerbate tau spreading in PS19 tauopathy mice, I crossed in the TMEM16F knockout allele. In a preliminary assessment, I found that PS19+ TMEM16F KO mice have decreased levels of both microgliosis and hyperphosphorylated tau. Thus, tau propagation is reduced by the removal of TMEM16F, which also reduces MVs, raising the possibility that MVs rather than exosomes play a major role in the spreading of tau. For the F99 phase of this fellowship, I propose to investigate the role of TMEM16F on tauopathy and the contribution of different EVs in the spreading of pathogenic tau. My experimental strategy combines: primary neuronal cultures and in vitro biosensor assays to characterize seeding propensity of microglial EV tau (Aim 2a), mouse behavioral assays to assess learning and memory impairments (Aim 2b), and immunohistochemistry to analyze pathology (Aim 2c). For the K00 phase of this fellowship, I propose to leverage the broad range of conceptual and technical expertise I have amassed studying microglial EV secretion in tauopathy to complete a postdoctoral fellowship studying microglial misregulation in neurodegeneration. Specifically, I will combine my graduate research experience with bioinformatics, flow cytometry, advanced microscopy, and in vivo models of neurodegeneration to provide new insight into neuroinflammation and immune receptor dysfunction in neurodegeneration and ultimately, uncover new therapeutic targets for neurodegenerative disease.